As a variety of eukaryotic cells age, the specific activity of glucose- 6-phosphate dehydrogenase (G-6-PDH) declines as much as 50%. Due to the central role of this enzyme in metabolism, it is important to define factors responsible for this loss in enzyme activity. One promising possibility is that this may be due to oxidative modification, a process which has recently been implicated in the age-related loss in activity of a number of enzymes. We have found that G-6-PDH from Leuconostoc mesenteroides is rapidly inactivated by micromolar concentrations of ferrous iron and hydrogen peroxide. Inactivation correlated with the formation of one carbonyl functionality per enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification. Our results suggest that ferrous iron binds to the glucose-6phosphate binding site and that interaction of the enzyme-bound ferrous iron with hydrogen peroxide leads to the oxidative modification of amino acids essential for enzyme activity. Partially inactivated enzyme remained predominantly in the dimeric form and no change in the apparent affinity of the remaining active subunits for substrate was observed. Partial inactivation did, however, lead to a decrease in the thermal stability of the remaining activity. This decrease in thermal stability could be largely overcome by the addition of glucose-6-phosphate. Thus, while exposure to hydrogen peroxide and ferrous iron results in the irreversible inactivation of G-6-PDH, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large-scale structural changes. Our results demonstrate the inherent susceptibility of G-6-PDH from L. mesenteroides to modification by an oxidation system known to exist in vivo. An assessment of the physiological significance of ferrous iron-catalyzed oxidation of G-6-PDH awaits extension of these studies to mammalian sources known to accumulate less active or inactive forms of the enzyme as a function of age.